One of the most useful approaches for studying the functions of specific genes (including their health related functions) is to examine the effects of mutations within those genes (i.e., the phenotype of the mutation). This approach involves correlating mutations within specific genes with the phenotypes or disease conditions that result from those mutations. This has been particularly fruitful in recent years with the identification of genes for such diseases as cystic fibrosis (Snouwaert et al., Science, 257:1083 (1992)), obesity (Zhang et al, Nature, 372: 425 (1994)), polycystic kidney disease (Moyer et al., Science, 264:1329 (1994)), breast cancer [Miki et al., Science, 266:66-71 (1994); Tavtigian et al., Nat. Genet., 12:333-337 (1996)], and other diseases. In these cases, the function of the implicated genes was not apparent solely from their DNA sequence but rather was defined by a disease condition associated with mutations in the genes.
A particularly productive approach to understanding the function of a particular gene in animals involves the disruption of the gene's function which is colloquially referred to as a "targeted mutagenesis". One common form of targeted mutagenesis is to generate "gene knockouts". Typically, a gene knockout involves disrupting a gene in the germline of an animal at an early embryonic stage. (See, Thomas et al., Cell, 51:503 (1987).) Once established in the germline, it is possible to determine the effect of the mutation on the animal in both the heterozygous and homozygous states by appropriate breeding of mice having the germline mutation.
Among the many examples of the use of knockout technology utilized to investigate gene function are U.S. Pat. Nos. 5,625,122 and 5,530,178 to Mak, T. which describe the production of mice having a disrupted gene encoding lymphocyte-specific tyrosine kinase p56.sup.lck and Lyt-2, respectively. Silva et al., Science, 257:201 (1992) produced mice having a disrupted .alpha.-Calcium Calmodulin kinase II gene (.alpha.CaMKII gene) which resulted in animals having an abnormal fear response and aggressive behavior. (See, also, Chen et al., Science, 266:291 [1994]). Wang et al., Science, 269:1108 (1995) demonstrated that the disruption in mice of the C/EPB.alpha. gene which encodes a basic leucine zipper transcription factor results in impaired energy homeostasis in the mutant animals. Knudsen et al., Science, 270:960 (1995) demonstrated that disruption of the BAX gene in mice results in lymphoid hyperplasia and male germ cell death.
The most common approach to producing knockout animals involves the disruption of a target gene by inserting into the target gene (usually in embryonic stem cells), via homologous recombination, a DNA construct encoding a selectable marker gene flanked by DNA sequences homologous to part of the target gene. When properly designed, the DNA construct effectively integrates into and disrupts the targeted gene thereby preventing expression of an active gene product encoded by that gene.
Homologous recombination involves recombination between two genetic elements (either extrachromosomally, intrachromosomally, or between an extrachromosomal element and a chromosomal locus) via homologous DNA sequences, which results in the physical exchange of DNA between the genetic element. Homologous recombination is not limited to mammalian cells but also occurs in bacterial cells, yeast cells, in the slime mold Dictyostelium discoideum and in other organisms. For a review of homologous recombination in mammalian cells, see Bollag et al., Ann. Rev. Genet., 23:199-225 (1989) (incorporated herein by reference). For a review of homologous recombination in fungal cells, see Orr-Weaver et al., Microbiol. Reviews, 49:33-58 (1985) incorporated herein by reference.
As is illustrated by the foregoing, gene knockout technology has often been used in mice and has allowed the identification of the function of numerous genes and, in some cases, ascertainment of their roles in disease. Much may be learned about the function of human genes from studies of mouse genetics because the vast majority of genes in humans have homologous counterparts in the mouse. Because of this high level of homology between the species, it is now possible to define the function of individual human genes and to elucidate their roles in health and disease by making targeted germline mutations in selected genes in the mouse. The phenotype of the resulting mutant mice can be used to help define the phenotype in humans.
With the increasing awareness that mouse mutations can provide such useful insights about the function of genes from humans, a great deal of interest is developing to systematically generate mutations within genes in mice that correspond to those genes which are being isolated and characterized as part of various genome initiatives such as the Human Genome Project. The problem with utilizing these procedures for large-scale mutagenesis experiments is that the technologies for generating transgenic animals and targeted mutations are currently very tedious, expensive, and labor intensive.
One of the biggest problems with the efficient generation of targeted mutations is the generation of the targeting construct. Targeting constructs are typically prepared by isolating genomic clones containing the region of interest, developing restriction maps, frequently engineering restriction sites into the clones, and manually cutting and pasting fragments to engineer the construct. See, e.g., Mak, T. U.S. Pat. Nos. 5,625,122 and 5,530,178; Joyner et al., Nature, 338:153-156 (1989); Thomas et al., supra; Silva et al., supra, Chen et al., supra; Wang et al., supra; and Knudsen et al., supra. This process can take a single highly skilled individual at least several weeks, often several months, to complete. Thus, in order to more rapidly and efficiently elucidate the functions of a variety of genes and to understand their role in health and disease, there exists a need to develop more efficient methods for the production of targeting constructs which do not require detailed restriction mapping and certain other complex molecular engineering steps.